Regenerative medicine holds great promise for the treatment of a wide range of diseases and major advances have been made in the ability to control and direct cellular differentiation for the purpose of tissue repair. However, the developmental signaling mechanisms are complex and cellular differentiation protocols in vitro and tissue regeneration in vivo require much improvement. Members of the TGF- superfamily including Activins, Nodal, Growth and Differentiation Factors (GDFs) and Bone Morphogenetic Proteins (BMPs) play crucial roles during development and tissue morphogenesis and have been widely used in regenerative medicine applications. The goal of the present study is to make structure-guided modifications of TGF- superfamily ligands in order to develop new signaling activities that enhance cellular differentiation steps required for effective regenerativ medicine applications in musculoskeletal disorders. Mesodermal specification is driven by Nodal signaling during natural vertebrate development and represents a critical early step in the differentiation of pluripotent progenitor cells toward many lineages including bone, tendon and cartilage. Current protocols for in vitro mesodermal differentiation use varying concentrations of Activin A and can likely be improved significantly as they neglect Nodal signaling altogether. Aim 1 of this proposal will generate chimeric Nodal and Activin molecules with a range of signaling activities that we will then use to fine tune mesodermal differentiation in vitro. The structural and mechanistic basis of Nodal and Activin complex assembly with their type I (ALK4) and type II (ActRII and ActRIIB) signaling receptors and the co-receptor Cripto will also be determined and the resulting information will be used to assist in engineering the chimeric ligands. Directed terminal differentiation of osteocytes, chondrocytes and tenocytes is critical fo regeneration of bone, cartilage and tendon, respectively. Current protocols for specifying these cell fates use high doses of specific BMPs and GDFs. Aim 2 will test the hypothesis that enhancing the potency of BMPs/GDFs by substituting their low affinity type II receptor binding epitopes for the high affinity binding epitope of Activin A will result in engineered ligands with heightened ability to promote terminal differentiation of osteo-, chondro- and tenocytes. It will also test if additional structure-guided modifications can further increase type I or type II receptor binding to make these ligands even more effective for this purpose. Overall, these studies will not only pave the way towards discovering potentially powerful therapeutics for musculoskeletal diseases but also elucidate key structural and mechanistic aspects of signaling of TGF- superfamily ligands.